ABSTRACT. Latent HIV infection within the CNS remains a major problem. Precisely identifying the latently infected cell types, the mechanisms of viral reactivation, and the approach for elimination of HIV-1 in the CNS have been the topics of much research over the years. CRISPR/Cas9 has shown promise in removing productively infect- ed cells from the periphery, but its penetration to and impact in the brain has yet to be investigated. The salient feature of this method lies in its ability to edit the viral genome and permanently inactivate it in the latently in- fected cells by removing a large fragment of viral DNA with no requirement for latent virus activa- tion/reawakening. The Long-Term Goal is to establish a basis for eradication of HIV from reservoirs, including the CNS. The Objective of this Application is to develop a strategy for, and a means of measuring effective- ness of, removing virus from latently infected cells using CRISPR/Cas9, without killing the infected cell. The Central Hypothesis is that removal of key stretches of integrated DNA from the host cell would render the virus replication incompetent, and that this would result in elimination of viral protein production, which has a neuro- toxic effect, i.e. Tat, gp120 and then secretion in the brain environment. This is based this on previous work from the interdisciplinary team of neuroimmunology (Khalili & MacLean), HIV-1 molecular geneticist (Khalili), glia activation in brains in the absence of viral replication (MacLean), experience with reactivation of latently in- fected T cells (Ling), and gene therapy based approaches to chronic diseases (Bunnell). We will employ CRISPR/Cas9 gene editing strategy to excise various regions of SIV-1 including the sequences between the 5'-LTR and gag, as well as the entire viral coding sequence located between the 5'- and 3'-LTRs. The Ra- tionale for these studies, is that once this project is completed, it will be possible to eradicate HIV from CNS reservoirs without causing deleterious neuroinflammation. Two Specific Aims are proposed: Specific Aim 1: Determine the phenotype of latently-infected cells in the CNS. Microglia and astrocytes are the major res- ervoirs for viral latency in the CNS. We hypothesize that latent infection (integrated DNA) of these cells results in irreversible activation or other phenotypic changes that can distinguish latently infected cells in the CNS. Our central hypothesis for this aim is that CRISPR/Cas9 gene editing will reduce or even reverse this activation with regard to innate immune activation, and expression of proinflammtory cytokines and viral proteins. Specif- ic Aim 2: Deliver CRISPR/Cas9 to the CNS using an adeno-associated viral delivery vector. Our hypothe- sis here is that AAV will facilitate delivery of CRISPR/Cas9 through the blood-brain barrier to latently infected cells following SIV infection, and by inference, HIV infection. Our central hypothesis is that the removal of longer sequences of virus will substantially reduce viral reservoirs, and the inflammatory profile of CNS cells in HIV infected individuals.